Protein immobilization has advanced significantly allowing for its application in a wide variety of areas including biosensors and nanotechnology. The process of immobilizing enzymes to solid supports provides unique capabilities for bioreactors, including the facile separation of enzyme from products and in some cases, increased enzyme stability and activity. These benefits can be realized to envision a high throughput technology for DNA sequencing, where the supported enzyme consists of lambda exonuclease, a highly processive enzyme that digests dsDNA into mononucleotides (dNMPs). Therefore, the basic hypothesis behind our sensor functionality is: Individual dNMPs moving electrokinetically through a 2D nanochannel will experience flight-times dependent upon the molecular identity of the dNMP, the chemical identity of the surface of the nanochannel and its dimensions. As a necessary step toward the realization of this DNA sequencing technology, the following aims must be achieved: 1.To generate solid-phase nano-reactors via thermal nano- imprint lithography (NIL) using polymer stamps consisting of taper inputs, entropic traps for DNA shaping and a single nano-pillar serving as a solid-support for the exonuclease enzyme. 2. Investigate the translocation of dsDNA through these irregular structures with feature sizes approaching the persistence length of dsDNAs, and 3. Immobilize enzymes to the solid support and monitor the digestion of single dsDNA molecules. Our preliminary results suggest that the immobilization of lambda exonuclease to a solid support can successfully be accomplished using 3-(3-dimethylaminopropyl) carbodiimide/ N- hydroxysuccinimide (EDC/NHS) coupling chemistry to a polymer support with micrometer dimensions that has been activated with UV light to produce a carboxylic acid functional scaffold. Fluorescence results from dsDNA digestion suggest lambda exonuclease was attached to the support and maintains its activity with increased activity compared to the same enzyme in solution. In this F31 grant application I will introduce the DNA sequencing technology we are exploring and the solid- phase bioreactors we are constructing using lambda exonuclease as the enzyme to disassemble the dsDNA molecule being interrogated. I will also discuss our results on producing silicon masters for the fabrication of our polymer nanopillars and methods that will be used to immobilize the enzyme to these nanopillars, as well discuss the plans for assessing the enzymatic cleavage of the double- stranded DNA in these nano-reactors and the planned translocation experiments of this DNA through the device design. PUBLIC HEALTH RELEVANCE: While genome sequencing has evolved into methods that offer higher throughputs that could assist with improving patient diagnoses, the ability to make the technology more accessible still remains a challenge. Accordingly, the project proposed here builds on this foundation by expanding accessibility to sequencing through the development of a DNA sequencing tool that is a combination of sequential DNA disassembly using immobilized exonuclease enzymes and flight-time-based identification of single DNA monomers through a nanochannel sensor made from plastics by means of low-cost replication. This project aims to make these technologies more affordable, while also increasing their throughput for ascertaining important data in a variety of critical application areas, including in vitro DNA diagnostics.